Cytochrome c is a key protein in the electron transfer system of eucaryotic mitochondria. Its amino acid sequence and structure are homologous after evolution time spanning more than a billion years. To investigate the role of individual amino acid residues on the structure and function, a method to replace an amino acid at any position of the amino acid sequence would be useful in substitution or labelling with isotope of individual amino acids. The method is a total synthesis of cytochrome c. As reported previously, a breakthrough came in an attempt of this synthesis when we found cytochrome c synthetase in yeast mitochondria. The enzyme catalyzes covalent bonding of heme to apocytochrome c through two thioether bonds. In the current year, we found that there are two cytochrome c syntheses in yeast mitochondria, one solubilized recognizing yeast and Candida krusei apocytochrome c and partially tuna apocytochrome c but not horse apocytochrome c and the other not solubilized, recognizing both yeast and horse apocytochromes c. We speculate that the latter less specific enzyme might catalyze also covalent attachment of heme to apocytochrome c1. We have also solubilized cytochrome c synthetase from beef heart mitochondria and that this solubilized enzyme can catalyze attachment of heme to either horse or yeast apocytochrome c. Thus, this enzyme may be used in synthesis of cytochrome c. As a first step of synthesis of horse cytochrome c apofragment containing residues 1 to 37 was synthesized using the Merrifield's improved solid phase method and is being purified to be tested as a substrate for cytochrome c synthetase. An undecapeptide containing residues 28 to 36 plus methonine and glycine as the penultimate and COOH-terminal residues, respectively was also synthesized to be linked to a semisynthetic apofragment of residues 38 to 104. A fragment of residues 66 to 104 is also being synthesized to be linked with fragment residues 28 to 65. These fragments would be the building blocks in the total synthesis of cytochrome c.